Since the first demonstration in 1988 that mitochondrial DNA (mtDNA) base substitution and deletion mutations are linked to human disease, a variety of degenerative diseases have been associated with mtDNA mutations (reviewed in Wallace, D. C.[1994]J. Bioenergetics and Biomembranes 26:241-250). For example, certain deleterious base substitutions can cause familial deafness and some cases of Alzheimer's disease and Parkinson's disease. Other nucleotide substitutions have been associated with Leber's Hereditary Optic Neuropathy (LHON) and myoclonic epilepsy and ragged-red fiber disease (MERF). Base substitutions can also cause pediatric diseases such as Leigh's syndrome and dystonia. Severe rearrangements involving deletions have been linked with adult-onset chronic progressive external opthalmoplegia (CPEO) and Kearns-Sayre syndrome (KSS) as well as the lethal childhood disorder Pearson's marrow/pancreas syndrome (Wallace [1994], supra).
Somatic gene therapy.
Three different approaches for somatic gene therapy (reviewed in Ledley, F. D. [1996] Pharmaceutical Res. 13:1996) can be distinguished based on the nature of the material that is administered to the patient: (a) cell-based approaches involving the administration to the patient of genetically engineered cells ("ex-vivo"), (b) administration to the patient of genetically engineered, attenuated, or defective viruses, and (c) plasmid-based approaches that involve pharmaceutical formulations of DNA molecules. A variety of viral and non-viral methods have been developed for introducing DNA molecules into a cell. Non-viral techniques include precipitation of DNA with calcium phosphate (Chen, C., H. Okayama [1987] Mol. Cell. Biol. 7:2745-2752), dextran derivatives (Sompayrac, L., K. Danna [1981]PNAS 12:7575-7584), or polybrene (Aubin, R. J., M. Weinfield, M. C. Paterson [1988] Somatic Cell Mol. Genet. 14:155-167); direct introduction of DNA using cell elctroporation (Neuman, E., M. Schaefer-Ridder, Y. Wang, P. H. Hofschneider [1982] EMBO J. 1:841-845) or DNA microinjection (Capecchi, M. R. [1980] Cell 22:479-486); complexation of DNA with polycations (Kabanov, A. V., V. A. Kabanov [1995] Bioconjugate Chem. 6:7-20); and DNA incorporation in reconstructed virus coats (Schreier, H., R. Chander, V. Weissig et al. [1992] Proceed. Intern. Symp. Control. Rel. Bioact. Mater. 19:70-71; Schreier, H., M. Ausborn, S. Gunther, V. Weissig, R. Chander [1995] J. Molecular Recog. 8:59-62).
Cationic lipids have become important reagents for gene transfer in vitro and in vivo. Several clinical trials approved by the NIH are in progress (reviewed in Ledley, F. D. [1994] Current Opinion in Biotechnology 5:626-636; and Ledley, F. D. [1995] Human Gene Therapy 6:1129-1144). In terms of transfection efficiency, virus-based vectors are superior to all other DNA transfection methods. Several different viral vectors have been developed and are in clinical trials including those derived from murine leukemia viruses (retroviruses), adeno-associated virus, and adenovirus (reviewed in Ledley [1996], supra).
Transfection of mitochondria.
There have been only a few reports of nucleic acids entering mitochondria, and most have focused on the nuclear encoded RNA component of the mitochondrial RNA processing activity, RNase MRP (Chang, D. D., D. A. Clayton [1987] Science 235:1178-1184; and Li, K., C. S. Smagulas, W. J. Parsons et al. [1994] J. Cell. Biol. 124:871-882). The uptake of exogenous DNA into mitochondria involving the protein import pathway has been reported from two laboratories. Vestweber and Schatz ([1989] Nature (London) 338:170-172) achieved uptake of a 24-bp both single- and double-stranded oligonucleotide into yeast mitochondria by coupling the 5' end of the oligonucleotide to a precursor protein consisting of the yeast cytochrome c oxidase subunit IV presequence fused to a modified mouse dihydrofolate reductase. More recently, Seibel et al. (1995, Nucleic Acids Research 23:10-17) reported the import into the mitochondrial matrix of double-stranded DNA molecules conjugated to the amino-terminal leader peptide of the rat ornithine-transcarbamylase. Both studies, however, were done with isolated mitochondria not addressing the question of how oligonucleotide-peptideconjugates will pass the cytosolic membrane and reach mitochondrial proximity. Negatively-charged biological cell surfaces and lysosomal degradation establish major obstacles which are very unlikely to be overcome by single oligonucleotide-peptide complexes.
Dequalinium.
Dequalinium (DQA) Babbs, M., H. O. J. Collier, W. C. Austin et al. [1955] J. Pharm. Pharmacol. 8:110-119) has been used for over 30 years as a topical antimicrobial agent. There is no consensus about the molecular target of DQA; several different targets such as the small conductance Ca.sup.2+ -activated K.sup.+ channel, F1-ATPase, calmodulin, and proteinase K have been suggested (Dunn, P. M. [1994] Eur. J. Pharmacology 252:189-194; Zhuo, S., W. S. Allison [1988] Biochem. Biophys. Res. Comm. 152:968-972; Bodden, W. L., S. P. Palayoor, W. N. Hait [1986] Biochem. Biophys. Res. Comm. 135:574-582; Rotenberg, S. A., S. Smiley, M. Ueffing et al. [1990] Cancer Res. 50:677-685). DQA is an amphiphilic dicationic compound resembling bolaform electrolytes, that is, they are symmetrical molecules with two charge centers separated at a relatively large distance. Lipophilic cations are known to localize in mitochondria of living cells as a result of the electric potential across the mitochondrial membrane (Johnson, L. V., M. L. Walsh, B. J. Bockus, L. B. Chen [1981] J. Cell. Biol. 88:526-535). The accumulation of DQA in mitochondria has been reported (Weiss, M. J., J. R. Wong, C. S. Ha et al. [1987] PNAS 84:5444-5448; Christman, E. J., D. S. Miller, P. Coward et al. [1990] Gynecol. Oncol. 39:72-79; Steichen, J. D., M. J. Weiss, D. R. Elmaleh, R. L. Martuza [1991] J. Neurosurg. 74:116-122; Vercesi, A. E., C. F. Bernardes, M. E. Hoffman et al. [1991] J. Biol. Chem. 266:14431-14434).
Despite the progress being made in developing viral and non-viral DNA delivery systems, there is a need for an efficient method for introducing DNA into mitochondria of intact cells.